We describe a novel method for determining serum triglycerides, in which an enzymatic hydrolysis replaces the more commonly used saponification procedure. Under the conditions of the assay, the enzymatic hydrolysis can be completed in less than 10 min by the combined action of a microbial lipase and a protease. We have been able to demonstrate complete hydrolysis of triglycerides by thin-layer chromatography of the reaction products, by recovery of glycerol from sera of known triglycerides content, and by comparison of triglyceride assays on a number of sera assayed by our method vs. the AutoAnalyzer procedure. The hydrolysis is directly coupled to the enzymatic determination of glycerol, and is followed through absorbance changes at 340 nm. The assay is simple, rapid, and requires only 50 µl or less of sample. Because the enzymes used do not release glycerol from other compounds in serum, the hydrolysis can be considered specific for triglycerides.

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A procedure for enzymatic determination of serum triglycerides [Clin. Chem. 19, 476 (1973)] has been adapted for use in continuous-flow analysis (Technicon AutoAnalyzer). A very simple manifold is used; serum is incubated at 37 °C with the lipase and α-chymotrypsin in potassium phosphate buffer (0.1 mol/liter, pH 7, containing 1.50 g of bovine serum albumin per liter). The liberated glycerol is dialyzed against the complete glycerol reagent. The change in absorbance at 340 nm resulting from oxidation of NADH is proportional to the dialyzed glycerol. The same manifold can be used to determine preformed glycerol if the hydrolyzing enzymes are omitted. The lipase used need not be extensively purified because the dialyzer removes coloring material and contaminating enzymes that would interfere with the components of the glycerol reagent in the manual procedure. The hydrolysis is complete, as shown by the use of equivalent glycerol standards. No prior treatment of the samples is necessary. Assays are run at 60 per hour in the AutoAnalyzer I, 80 per hour in the AutoAnalyzer II. Results with both instruments for 150 samples correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure.

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We describe a kinetic method for assay of serum or plasma triglycerides, by use of an enzymatic hydrolysis and reaction sequence already described [Clin. Chem. 19, 476 (1973)]. The reaction is triggered by addition of lipase, at a time when free glycerol, or pyruvate (or both) are no longer present. In this method, therefore, there is no need for a blank glycerol assay. In the procedure, reagents are used that are available commercially in the form of stable, dry powders; the method for the preparation of the reagents has been changed to achieve improved stability and performance. Stability and recovery of added triglycerides are satisfactory.

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